Retinal ganglion cell (RGC) loss is the primary contributor to visual loss in glaucoma. Recent evidence shows that apoptosis contributes to RGC loss. A wide variety of insults can initiate neuronal apoptosis via different signaling pathways. The initiating insults and signaling pathways that operate in glaucoma are not understood. Thus the overall objective of this proposal is to determine the intracellular signal path resulting in the death of RGC in four different rat models; chronic high IOP, retinal ischemia, retinal excitotoxicity and trophic withdrawal. We will use laser confocal scanning microscopy techniques for retinal flatmounts that allow cells within specific retinal layers to be simultaneously imaged at high subcellular resolution for nucleic acid markers, markers for specific organelles and immunofluorescence for antibodies to specific cellular proteins. These methods will be employed in the above rat models of RGC death to determine apoptotic signalling proteins. Aim 1 is to determine the presence of markers for apoptosis in the cell nuclei of RGC. These markers are a) histone re-organization, b) poly ADP-ribosylation of nuclear proteins, c) increased expression and nuclear binding of transcription factor p53, d) apoptotic DNA cleavage and nuclear condensation, e) translocation of glyceraldehyde phosphate dehydrogenase to the nucleus. Aim 2 is to determine mitochondrial markers for apoptosis in stressed RGC including a) reduced mitochondrial membrane potential, b) increases in expression and mitochondrial binding of the pro-apoptotic signal protein BAX and decreases for the anti-apoptotic signal protein BCL-XL. Aim 3 is to determine the level of additional intracellular apoptotic markers in stressed RGC including the activation of caspases 3,9,2 and the anti-apoptotic kinases phospho AKT and phosphatidylinositol phosphate kinase 3. The results of experiments will allow identification of apoptotic pathways in RGC associated with specific stresses/insults to the retina, and the possibility of entirely new therapeutic interventions for retinopathies.